Our recent work has demonstrated that the immunosuppressive drug rapamycin selectively affects the CsA-resistant pathway. Our initial studies have focused on the mechanism of activation of the IL-2 gene in a CsA-resistant manner. We found that the effect of rapamycin on the IL-2 expression was due to alteration in IL-2 mRNA stability. More recently, we have also shown that activation of T cells by IL-12 is resistant to CsA, but sensitive to rapamycin. As the intracellular target of rapamycin is mTOR (mammalian target of rapamycin), we are investigating the mechanism of activation of mTOR during T cell activation. Our preliminary data indicate that PKC-theta plays an important role in turning on mTOR downstream signaling. Regarding the physiological role of the resistant pathway, we have observed the effect of cytokine signaling, particularly the combination of IL-12 and IL-18 but not individual cytokine alone, in activating resting human peripheral blood T cells in producing IFN-g in a CsA-resistant, but rapamycin-sensitive manner. Bothe nave and memory CD4+ T cells are responsive to IL-12 plus IL-18 stimulation. This cytokine-mediated activation of resting T cells is independent of antigen. Intracellular staining reveals a small percentage of resting CD4+ T cells capable of producing IFN-g in response to IL-12 plus IL-18. We have shown that CD4+IL-18Ra+ cells are the main populations responsive to IL-12 plus IL-18. The expression of IFN-g induced by IL-12 and IL-18 is sensitive to rapamycin and SB203580, indicating the possible involvement of mTOR and p38 MAP kinase, respectively, in this synergistic pathway. We have also shown that IL-12 plus IL-18-induced IFN-g mRNA expression is independent of new protein synthesis and independent of GADD45b and g. We are currently investigating the in vivo physiological role of this cytokine signaling pathway.